The invention relates to a process and a composition for determining the activity of creatinekinase-MB in human body fluids.
The determination of the activity of creatinekinase (ATP: creatine-phosphotransferase, E.C. 2.7.3.2; abbreviation; CK) in serum is considered the most sensitive laboratory method for diagnosing diseases of skeletal muscles and the myocardium, especially myocardial infarction. However, differentiation between trauma of skeletal muscles and the myocardium is difficult, especially in making a differential diagnosis of myocardial infarction. Determination of total CK activity results in unreliable differentiation.
Prior attempts to improve the reliability of evidence provided by determination of CK activity in differential diagnosis have used measurements of activity of other enzymes in the serum and correlation of the resulting measurement with each other, for example, quotient CK/glutamic oxalacetic transaminase. However quotients of this type cannot be used to differentiate between cardiac infarction and pulmonary infarction or between cardiac infarction and secondary shock resulting from other causes.
CK occurs in the body in the form of three isoenzymes, namely CK-MM, for example, in muscles; CK-BB, for example, in the brain; and hybrid CK-MB, consisting of an M and a B subunit, for example, in the myocardium. CK activity in blood serum is normally due to the CK-MM isoenzyme, because CK-BB does not pass through the fluid-blood barrier and CK-MB is restricted to certain organs, for example, the myocardium. However, when the myocardium is damaged, as in cardiac infarction, CK-MB is released into the blood serum and can be detected there.
Quantitative determination of this isoenzyme along with CK-MM in the serum is considered the most sensitive laboratory method and provides the greatest evidence in differential diagnosis of cardiac infarction. It is true that CK-MB is present in other organs, for example, the pancreas, the diaphragm, the aorta, the lungs and the uterus, as well as in the myocardium but the activity thereof in these organs is about 100 times less than in the myocardium, so that any CK-MB activity liberated from these other organs is below the limits of detection.
Determination of CK-MB activity previously was based essentially on one of three methods:
1. Electrophoresis on various carriers. The results obtained by this method are sometimes contradictory. Frequently the number of bands appearing is greater than the number of isoenzymes present, that is, artifacts cause unreliability.
2. Column chromatography on various materials. This method is time-consuming, has an actual operating time of several hours and is therefore not suitable for routine investigations. Results obtained by different investigators are sometimes contradictory.
3. Immunological determination by antibodies causing precipitation. This method, described in German Pat. Application Nos. P 21 28 670, (U.S. Pat. No. 3,932,221), and P 22 58 822, (U.S. Ser. No. 419,283, filed Nov. 27, 1973) gives good results, for example, in the quantitative determination of aldolase and alkaline phosphatase isoenzymes. However, the sensitivity of the method is inadequate for determination of relatively low activities of CK and especially of CK-MB.
In the processes described in German Pat. applications Nos. P 21 28 670 and P 22 58 822, the isoenzymes of CK are precipitated by antibodies which effect precipitation and act specifically on isoenzymes. In each case the residual activity in the supernatant liquor from the immune precipitation is determined. Apart from the effort involved in this method, which as a rule necessitates the preparation of several specific antisera, it is necessary to carry out at least two different tests, that is, determination of total CK activity and determination of residual CK activity after precipitation. Thus, the CK-MB activity can be determined only by measuring a difference. The result is therefore subject, in accordance with the rules of the theory of errors, to the uncertainty of both measurements. In the method of the invention, the addition of errors is avoided by making a direct measurement.
Another disadvantage of the precipitation process is that the immune precipitation, which is a secondary reaction, takes from about 60 minutes to several hours, so that the process is not suitable as a rapid test.
Owing to the relatively long time required to carry out all of the above processes, they are not suitable for rapid diagnosis of cardiac infarction.
Thus, there is a continuing need for a simple, rapid and reproducible process and composition for determining the activity of CK-MB in a sample of a body fluid.
This object is achieved according to the invention by a process which operates by using specific antibodies to completely inhibit enzymatic activity of the M subunit in CK-MM and CK-MB without inactivating enzymatic activity of the B subunit in any CK-MB which may be present.